Altered regulation of lipid and glucose homeostasis, most often in the setting of insulin resistance and obesity, is central to the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Because current management options remain limited, the discovery of new metabolic pathways will serve to identify novel opportunities for pharmacologic intervention. This research proposal addresses the unanswered question of whether membrane phospholipids regulate nutrient homeostasis. Our long-term goal is to understand how phospholipid-mediated metabolic control can be leveraged for therapeutic purposes. The objective of this research is to determine the molecular mechanisms whereby sensing of membrane phosphatidylcholines (PC) by phosphatidylcholine transfer protein (PC-TP) is translated into metabolic control by thioesterase superfamily member 2 (Them2), an acyl-CoA thioesterase. The central hypothesis is that key regulatory events occur in the liver when PC-TP binds specific membrane PC molecular species and then activates Them2. The rationale is that the mechanisms of a PC-sensing pathway should yield new insights into hepatic insulin resistance and steatosis. Guided by extensive preliminary data, the central hypothesis will be tested in three specific aims: 1) To demonstrate that Them2 and PC-TP limit insulin signaling in liver by regulating glycerolipid production; 2) To elucidate the mechanisms by which Them2 and PC-TP promote hepatic insulin resistance and steatosis in response to overnutrition; and 3) To define the influence of ligand binding on the activation of Them2 by PC-TP. In Aim 1, mouse models will be used to determine a role for Them2 downstream of PC-TP in the production of diacylglycerols (DAG), which reduce insulin sensitivity by activating the novel protein kinase C isoform PKC?. Additional mechanistic insights will be gleaned from systematic studies in cell culture systems that relate Them2-mediated control of DAG production to insulin signaling through PKC?. Aim 2 will establish the roles of Them2 and PC-TP in hepatic insulin resistance and steatosis due to overproduction of DAG, PC and triacylglycerols, which lead to PKC? activation, endoplasmic reticulum (ER) stress and lipid accumulation in response to high fat feeding. Cell culture and in vitro studies will determine whether ER stress is attributable to impaired calcium homeostasis resulting from altered membrane composition. In Aim 3, control of Them2-PC-TP interactions by individual PC species will be quantified by pulldown assays, surface plasmon resonance, x-ray crystallography and the activity of Them2. The interacting domains of Them2 and PC-TP will be identified by hydrogen-deuterium exchange and by mutational analysis employing cell-based assay systems. Overall, this proposal will elucidate new mechanisms of phospholipid-mediated metabolic regulation in the liver, which is significant because the fatty acyl composition of the membrane PC varies in health and disease. These studies are expected to identify new therapeutic targets for the management of for NAFLD and associated disorders.